Microglia mediate neurocognitive deficits by eliminating C1q-tagged synapses in sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is a severe and frequent complication of sepsis causing delirium, coma, and long-term cognitive dysfunction. We identified microglia and C1q complement activation in hippocampal autopsy tissue of patients with sepsis and increased C1q-mediated synaptic pruning in a murine polymicrobial sepsis model. Unbiased transcriptomics of hippocampal tissue and isolated microglia derived from septic mice revealed an involvement of the innate immune system, complement activation, and up-regulation of lysosomal pathways during SAE in parallel to neuronal and synaptic damage. Microglial engulfment of C1q-tagged synapses could be prevented by stereotactic intrahippocampal injection of a specific C1q-blocking antibody. Pharmacologically targeting microglia by PLX5622, a CSF1-R inhibitor, reduced C1q levels and the number of C1q-tagged synapses, protected from neuronal damage and synapse loss, and improved neurocognitive outcome. Thus, we identified complement-dependent synaptic pruning by microglia as a crucial pathomechanism for the development of neuronal defects during SAE.


Novel object recognition test
At day 10 and 30 after PCI, the novel object recognition test (NOR) was performed as previously described (64). A black open field box (40 cm), illuminated with 15 Lux via LEDs, was used for the experiment which were performed by a blinded experimenter. Objects were clearly distinguishable and different for each testing day: an iron bracket and an objective container (both day 10), a small plastic cylinder, and an iron eyebolt (both day 30). The objects were previously tested and no preferences for an individual object could be found. Additionally, each object was randomized to be used as familiar or unknown object. Between each trial, the box was carefully cleaned with 70% ethanol to ensure equal experimental conditions. For habituation, mice were placed in the open field box for 10 minutes (5 min. for day 30) one day prior to the NOR. In the familiarization session on the following day, mice were allowed to explore two equal objects for maximum 10 minutes. Every touching of the object and facing an object in a distance of maximum 2 cm from the object was regarded as exploration behavior.
Climbing onto the object was not regarded as exploration behavior. The familiarization phase was terminated by the investigator after mice had explored the two objects for cumulative 20 seconds.
During the familiarization phase also the "time to reach criterion" was analyzed, meaning the time to reach 20 seconds at the objects to test any restrictions of locomotor or exploratory behavior. In the testing phase, six hours after the training phase, one object was replaced and mice were allowed to explore the familiar and unknown object. Exploration time was measured, and the session was completed after 20 seconds cumulative exploration behavior. Mice not exploring the objects for 20 seconds within 10 minutes in the familiarization or testing phase were excluded from the analysis.

Barnes Maze test
A circular white PVC platform (diameter 120 cm) was mounted on a rotatable stand, elevated 100 cm above floor level, and consistently illuminated by 2 light sources (900-1000 Lux).
The maze comprised 40 pseudo-randomly distributed and equally sized holes (diameter each 5 cm), 39 holes were closed with black PVC. During the habituation (d31 after PCI) and testing phase (d32-37 after PCI) an escape box was mounted below one of the holes. Four different holes at the respective sides of the maze were used as escape holes. The location of the escape box was kept constant for each mouse but varied between mice (total of 4 possible target locations). Three different black-colored geometric shapes printed on paper were used as cues and placed at the three walls surrounding the maze. At the open side of the maze the examiner was regarded as the cue. Before starting each experiment, mice were acclimated to the testing room for at least 30 minutes and each mouse was placed in an individual holding cage after the experiment. After each trial the maze was cleaned with 70% ethanol to ensure comparable experimental conditions. In the habituation phase (d31), mice were placed in a transparent plastic located in the center of the maze.
After 60 seconds the cylinder was lifted and mice were allowed to explore the maze for 60 seconds and then gently led to the escape box using the transparent cylinder. Mice were allowed to spend 2 minutes in the escape box before they were placed in individual holding cages. Each mouse underwent the habituation phase thrice with an inter-trial interval of 10 minutes. In the testing phase (d32-37), an opaque instead of the transparent plastic cylinder was used. The cylinder was lifted after 20 seconds and mice were videotaped and tracked (EthoVision) for maximum 4 minutes or until the mouse entered the escape box. Each mouse underwent the testing phase thrice with an inter-trial interval of 10 minutes. The mean latency until entry of the escape hole per day was calculated and used for further analysis (54).

Cell isolation
CD11b + primary microglia were isolated in all groups using MACS Technology (Miltenyi Biotec) according to manufacturer's instructions. Following preparation, brain samples were minced into small pieces and further dissociated enzymatically by Adult Brain Dissociation Kit (Miltenyi Biotec) and mechanically by gentle MACS Dissociator using program 37C_ABDK_01.
A single-cell suspension was obtained after a separation by a 70µm cell strainer. Cell suspension was labelled magnetically using CD11b-MicroBeads (Miltenyi Biotec) and loaded onto a MACS column for positive separation. Cells were immediately lysed in 250µl QIAzol (Qiagen) for further transcriptomic analysis.

RNA purification
Total RNA extraction was carried out using QIAzol lysis reagent and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer instructions; quality control of isolated RNA was performed on a QIAxcel capillary electrophoresis system (Qiagen, Hilden, Germany).
Integrity of RNA was proven by reporting the 8S/18S ratio and the RIS number (RNA Integrity Score) for analyzed samples. Quantification of total RNA and measuring the A260/A280 ratio and 260/230 nm were performed on a Nano-Drop spectrophotometer ND-2000 (Thermo Fisher Scientific, Schwerte, Germany).

RNA sequencing
Sequencing of RNA samples was performed using Illumina's next-generation sequencing methodology (65). In detail, total RNA (extraction see above) was quantified and quality checked using the Agilent 2100 Bioanalyzer Instrument in combination with RNA 6000 nano kit (both

FACS analysis
Transcardial perfusion with cold PBS (5ml/min, 4°C) for 2 min was performed to remove blood. Following brain removal, both hemispheres and the cerebellum were minced and subsequently digested enzymatically with collagenase, DNAse and trypsin-inhibitor for 45min at 37°C. Nondissociated brain tissue aggregates were removed using a 40 µm cell strainer. Cells were separated by standard density gradient centrifugation using Lymphoprep (Stemcell Technologies, Köln, Germany) and then centrifuged at 800g for 16min. Following centrifugation, the cell pellet was resuspended in FACS-buffer (PBS with 0.5% BSA, 2 mM EDTA and 0.1% sodium azide) for further analysis.
The protein concentration was determined using Bradford assay (BioRad Quick Start Bradford 1x Dye Reagent, #5000205) prior to protein analysis by capillary western immunoassay (WesTM, ProteinSimple) according to the user manual. For each target, the antibody dilution and dynamic range were determined independently to assure quantitative measurements (Table S3)

Measurement of serum neurofilament, UCH-L1 and tau protein
NfL assay by Quanterix HD-X was used to measure neurofilament light chain (Nfl) levels.
All available serum samples were analyzed for concentrations of Nfl, glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and tau protein in pg/ml using a single molecule array technique with a commercially available 4-plex kit (Neurology 4-plex kit, Quanterix) on a HD-X platform. Results were quantified using calibration curves with known standards assayed in the same run. Assays were done according to the manufacturers protocols except for analyzing singlicates (due to limited amount of serum) instead of duplicates after determining inter-and intra-assay coefficient of variance for Nfl/GFAP high and low controls to be below 10 %. The analysis was performed by blinded experimenters.
We performed inter-assay, intra-assay comparisons and precision measurements as well as inter-laboratory comparisons of Nfl and GFAP (Table S4) and identified a very low coefficient of variance in these parameters and excellent inter-laboratory correlations (Pearson correlation coefficient 0,9986) as described elsewhere (70).

Stereotactic intrahippocampal injection of antibodies
C1q blocking antibody (4.5mg/ml in PBS) and control antibody without specific antineuronal reactivity (purified patient IgG fractions, 4.5 mg/ml) were injected at 3 injection sites per hemisphere (Table S5) into the hippocampus 2 days prior to sepsis induction. were pulled with a micropipette puller (P-1000, Sutter) and cut with fine scissors to a diameter of several micrometers. After the injection finished, the injection pipette was carefully removed after a latency of 1-2 minutes and the surgical incision was closed using 2-3 michel suture clips (#BN507R, BBraun, Germany). Animals were monitored until they completely recovered from anesthesia.

Immunohistochemistry (mouse tissue)
To obtain mouse brain slices for immunohistochemistry, mice were deeply anesthetized with isoflurane and perfused with PBS after cardiac puncture. After preparation, brains were fixated for 24 h in 4% PFA, and dehydrated for 24 h in 10% and 24 h in 30% sucrose solution.
Thereafter, free-floating serial sections (16 µm and 40 μm respectively) were prepared. Slices were blocked with BB 2 (3% serum, 2% milk powder and 0.1% Triton X-100 in TRIS buffered saline For representative imaging of hippocampal C1q-labeled synapses engulfed in lysosomal microglial compartments, images were acquired on LSM900 (63x oil objective, NA = 1.4) using Airyscan mode. For this purpose, murine brain sections at d10 after PCI were stained for iba1, Homer1, CD68 as well as C1q. z-stacks were acquired with a 2028 x 2028 image resolution and with 8 pixel-wise plane scan intensity averages for each color channel and a 7.8 µm z-interval. To visualize C1q-dependent synapse elimination, representative microglia were selected and reconstructed in Imaris. For this purpose, the surface, the spot as well as the colocalization function was used for 3D reconstruction. C1q labeling of postsynaptic Homer1 was visualized using the "spots" function.
To validate specific binding of the C1q blocking antibody and to evaluate potential antibody diffusion after injection to the contralateral hemisphere, 16 µM thick tissue slices were stained against C1q ( in addition with a secondary antibody targeting mouse IgGs). Images were acquired with LSM900 (63x oil objective, NA = 1.4) using the Airyscan mode.
To quantify synaptic spots 10µm thick cryo slices stained with Homer1 and DAPI were imaged at two different locations in the CA1 region (8 images/mouse, 5-6 mice/group) on a Zeiss Elyra 7 lattice-SIM (63x oil objective, NA = 1.4). Images were acquired using the SIM function with a 1024 x 1024 image resolution. Scans were processed using Zen Software and Imaris.
Synapse quantification was performed with Imaris (surface function)

Immunohistochemistry (human tissue)
Three-micrometer thick sections from formalin-fixed paraffin-embedded autopsy brain tissue blocks were cut and processed for immunohistochemistry using standard routine procedures.
Tissue sections were stained with hematoxylin and eosin (H&E) and additional antibodies against Data are presented as mean ± SEM. Each circle represents one mouse.  PCI: n = 18; two-tailed Student's t-test) following sepsis induction.
Data are presented as mean ± SEM. Grey shading (in A, B, C) indicates human data.   Data are presented as mean ± SEM.  Data are presented as mean ± SEM. Data are presented as mean ± SEM.